哈萨克族食管鳞癌相关基因THSD7A的下游驱动基因筛选、临床意义和功能研究

As a malignant tumor, the esophageal cancer is aggressive and very poor prognosis. However, the molecular mechanism of etiology and pathogenesis is unclear now. In order to find the pathogenic gene, we use the second generation sequencing technology-sequencing the whole exome of Esophageal Squamous Cell Carcinoma(ESCC) in the Kazak and Uyghur ethnic. We have found that the mutation rate of THSD7A gene in Kazak ethnic(18.5%) is significantly higher than that in the Uyghur ethnic, which has racial differences. Further experiments on gene expression and function showed that THSD7A is associated with esophageal cancer and may be carcinogenic gene in ESCC. And more in-depth high-throughput chip experiments showed that THSD7A plays a role by several classical pathways and downstream genes. On the basis of previous research, this project is intended to carry out the verification and screening of new THSD7A downstream genes through a variety of mature experimental techniques(Clone formation experiment, RT-PCR, Western blotting, et al) from three aspects, in carcinoma tissue, cell and animal model. We want to analysis of the relationship between new downstream gene expression and malignant phenotype of Kazakh ESCC, and clarify its function in the occurrence and development of ESCC. So as to reveal the clinical significance and molecular function of the downstream driving gene, to lay the foundation for molecular classification and individual treatment of Kazakh ESCC.

食管癌侵袭性强且预后极差，目前其发生发展的分子生物学机制仍不清楚。为研究其潜在致病因子，课题组采用外显子二代测序，首次发现凝血酶敏感蛋白1型7A基因（THSD7A）在哈萨克族食管鳞癌组织中突变率高达18.5%，明显高于同地区的维吾尔族。进一步的基因表达和功能实验显示，THSD7A与哈萨克族食管癌相关且可能是食管鳞癌的致癌基因。更深入的高通量芯片实验显示，THSD7A通过多个经典通路和下游新基因发挥作用。在前期研究基础上，本项目拟开展疑似THSD7A下游新基因的验证、筛选，进而在哈萨克族食管鳞癌组织、细胞和动物实验三个层面，运用克隆形成、RT-PCR、Western blotting等成熟实验技术，发现下游驱动基因，分析下游新基因的表达与哈族食管鳞癌恶性表型的关系，明确其在食管癌发生、发展中的功能，揭示下游驱动基因的临床意义和分子生物学功能，为哈族食管癌分子分型和个体化靶向治疗奠定基础。

前期研究中，团队发现凝血酶敏感蛋白1型7A基因（THSD7A）是哈萨克族食管鳞癌（ESCC）致癌基因，且通过多个经典通路和下游新基因发挥作用。在此基础上，本项目进一步采用微阵列基因芯片实验、生物信息学分析、高通量Celigo增殖筛选实验及 qRT-PCR 等方法，初步筛选及验证了THSD7A下游ESCC相关潜在功能基因。研究团队发现SCARA5、ELF4、AP1S3是THSD7A下游可显著影响ESCC细胞增殖的潜在功能基因，WISP2则是ESCC中高表达且与患者临床恶性表型及不良预后相关的基因。进而，研究团队从细胞、动物、组织层面对潜在功能基因进行了临床意义和功能研究。研究结果显示：（1）SCARA5敲减后ESCC细胞的克隆形成能力、增殖能力、凋亡能力、侵袭迁移能力和裸鼠皮下成瘤能力均显著减弱，细胞周期被阻滞，与对照组相比有统计学差异（P＜0.05）；SCARA5在ESCC组织中较癌旁正常组织高表达（P＜0.05），但与患者临床病理特征与预后不相关（P＞0.05）；野生型与突变型SCARA5功能一致；（2）ELF4敲减后ESCC细胞的克隆形成能力、增殖能力、凋亡能力、侵袭迁移能力和裸鼠皮下成瘤能力均显著减弱，细胞周期被阻滞（P＜0.05）；ELF4在ESCC组织中较癌旁正常组织高表达（P＜0.05），并与患者临床分期、淋巴结转移和脉管浸润相关（P＜0.05），但与预后不相关（P＞0.05）；（3）AP1S3敲减后ESCC细胞的克隆形成能力、增殖能力、凋亡能力和侵袭迁移能力均显著减弱，细胞周期被阻滞（P＜0.05）；AP1S3在ESCC组织中较癌旁正常组织高表达（P＜0.05），并与患者临床分期、淋巴结转移、脉管浸润和不良预后相关（P＜0.05）；miR-204-5p可靶向下调AP1S3的表达水平，抑制ESCC细胞的增殖、迁移及侵袭能力；同时有可能通过下调PI3K/AKt通路蛋白磷酸化水平，抑制ESCC的EMT进程。（4）ESCC癌组织中WISP2较癌旁正常组织高表达，且与患者临床恶性表型及不良预后相关。总之，SCARA5、ELF4和AP1S3均为THSD7A下游ESCC相关促癌基因，或可与THSD7A联合应用于食管癌的精准个体化诊疗。WISP2或可作为ESCC新的预后指标和治疗靶点。